1. Field of the Invention
The invention relates to the use of nuclear magnetic resonance spectroscopy (NMR) to measure the utilization of one or more substrates in cells and tissues. In particular aspects the invention concerns the selection and use of .sup.13 C-enriched substrate(s) which are ultimately metabolized to acetyl-CoA. This method concerns the analysis of a single NMR spectrum which may be acquired at a time when steady-state isotopic or metabolic conditions may not have been established. The information obtained from this analysis is the fraction of acetyl CoA derived from the .sup.13 C-enriched substrate or substrates, and from unlabeled sources.
2. Description of Related Art
The relative rates of utilization of various exogenous and/or endogenous substrates in normal cells and tissues may be sensitive to many factors including cellular work rate, physiologic state, drugs, toxins, hormones, and the like. Substrate utilization may also be sensitive to disease states such as ischemia, infection, inflammation, trauma, congenital defects in metabolism, acquired defects in metabolism, or during malignant transformations such as cancer. Thus, precise quantitation of substrate utilization could have broad application since it may provide insight into the integrated functional state and viability of cells or a tissue.
Acetyl Coenzyme A (acetyl-CoA) is a key intermediate in cellular biochemistry. It is oxidized in the citric acid cycle for the production of energy, and it is a precursor in multiple biosynthetic processes. Acetyl-CoA may be derived from numerous compounds, each of which must be metabolized through different pathways subject to complex and interacting regulatory processes. Thus, the relative contribution of one or more substrates to acetyl-CoA reflects cellular metabolic state. Since this measurement is so important for the understanding of tissue metabolism, it has been the objective of numerous studies in many cellular preparations and tissues (see references 1-7 for examples using heart tissue).
The measurement of the contribution of a compound to acetyl-CoA ordinarily requires an estimate of the rate of acetyl-CoA utilization, typically from oxygen consumption, and the rate of substrate utilization under steady-state conditions. The latter usually is measured by the rate of appearance of .sup.14 CO.sub.2 from a .sup.14 C-enriched substrate, the rate of removal of substrate from the perfusion medium, or multiexponential analysis of .sup.11 C time-activity curves in tissues utilizing .sup.11 C-enriched substrates.
However, substrate and oxygen removal are difficult to measure under some important conditions, and metabolic and isotopic steady-state often cannot be assured. Further, since pyruvate may be metabolized by either pyruvate dehydrogenase or through a pyruvate carboxylation pathway the appearance of .sup.14 CO.sub.2 from .sup.14 C-enriched pyruvate (or its precursors) indicates net substrate oxidation only if the carbon skeleton enters the citric acid cycle via pyruvate dehydrogenase (8,9) Similarly .sup.14 CO.sub.2 release from fatty acids is an unreliable measure of this oxidation (10,11). Thus, standard methods for assessing substrate competition and oxidation are often not satisfactory for rapidly changing or spatially heterogeneous metabolic states, or if more than one pathway is available for carbon flow into the citric acid cycle.
In spite of these limitations on traditional methods, there is substantial interest in the measurement of substrate oxidation for the assessment of tissue metabolism and viability. For example, position emission tomography (PET) has been used to examine regional myocardial metabolism during ischemic and other states. However, the interpretation of some PET observations is controversial, for example, fatty acid oxidation in ischemic reperfused myocardium. PET studies generally have concluded that fatty acid oxidation is suppressed, but other reports have not validated this finding (4-7,12,13). PET is fundamentally limited by the lack of knowledge of the chemical state of the tracer. For example, a compound may enter a cell where it may be trapped and stored, metabolized to acetyl-CoA and oxidized, or it may remain in the cell briefly and then diffuse out, unchanged. Numerous assumptions regarding the metabolic fate of a tracer are therefore required.
For these reasons, some recent PET studies have emphasized the utilization of a very simple compound, acetate, which is not subject to many of the complex physiological processes which regulate normal metabolism (14). Analysis of the results is thereby simplified, but acetate is not a physiological substrate. Biochemical and physiological studies using .sup.11 C are also limited by the problem of working with a radioactive element with a very short half-life. Thus, a nearby cyclotron is essential, and rapid chemical synthesis is required. The study of some molecules or certain labeling patterns is simply not practical.
The analogous use of .sup.13 C enriched substrates to monitor intermediary metabolism has been established (15). Multiple enriched intermediates of the citric acid cycle may be detected by NMR spectroscopy (15-19). It has been shown that citric acid cycle flux may be determined if the fractional enrichments in intermediates are measured repeatedly after the addition of enriched substrate (17). This method, however, assumes steady-state flux conditions, constant intermediate pool sizes, and good temporal resolution. Although collection of in vivo data is theoretically possible, the method depends on measurement of fractional enrichment in glutamate and other intermediates, a requirement which may be difficult to meet under many important conditions.
An alternative to the measurement of absolute citric acid cycle flux is the measurement of the relative rates of competing pathways feeding acetyl-CoA. This approach has been reported previously. In some instances, metabolic and isotopic steady-state were assumed for the purposes of data analysis and these conditions were established experimentally (20,21). Other reports indicated that insight into the pathways feeding acetyl-CoA could be obtained by .sup.13 C NMR spectroscopy (18,19). Finally, one report described how to measure the ratio of the contribution of two labeled substrates to acetyl-CoA under nonsteady-state conditions (22). This method is limited, however, in that it requires that two different labeled substrates be administered, and the fraction of acetyl-CoA derived from unlabeled sources is not determined. Thus, methods for the measurement of the fraction of acetyl-CoA derived from a particular substrate or substrates under nonsteady state conditions have not been reported.
.sup.13 C NMR is useful for the monitoring of metabolism of .sup.13 C labeled compounds in experimental animals and humans. However, there are three important factors limiting study of substrate utilization in vivo. First, there is the consideration of expense. Significant amounts of relatively expensive labeled compounds make it difficult to maintain a constant concentration in the blood for the time required to attain isotopic steadystate. Second, many conditions of interest may involve rapidly changing metabolic conditions, and metabolic state cannot be assumed. Finally, an isotopomer analysis applicable in vivo to determine .sup.13 C contributions to the carbon skeleton of citric acid cycle components has been applied only when B.sub.0 homogeneity was sufficient to allow resolution of .sup.13 C--.sup.13 C scalar coupling. This condition of homogeneity is unlikely to be the case under the circumstances of many in vivo measurements.
The present invention addresses the problem of nonsteady-state conditions and field homogeneity requirements. It is shown that the contribution of one or more exogenously administered .sup.13 C-labeled substrates to acetyl CoA can be determined in a tissue or cell using .sup.13 C NMR without the constraint of metabolic or isotopic steady-state. Furthermore, the method permits the determination even when spectral lines are broad due to B.sub.0 inhomogeneity, thereby opening the way for substrate utilization studies in vivo. The method does not require many of the simplifying assumptions involved in .sup.11 C or .sup.14 C methods, and, since a stable isotope, .sup.13 C, is used, a wide variety of compounds with complex labeling patterns may be synthesized and studied.